Oncogene-blocking therapies: new insights from conditional mouse tumor models.

Identification of oncogene dependent signaling pathways controlling aggressive tumor growth has led to the emergence of a new era of oncogene-blocking therapies, including Herceptin and Gleevec. In the recent years conditional mouse tumor models have been established that allow switching-off the expression of specific oncogenes controlling tumor growth. The results may have two important implications for oncogene-blocking therapies: (i) downregulation of oncogenes, for instance HER2, MYC, RAS, RAF, BCR-ABL or WNT1, usually leads to a rapid tumor remission. However, it was observed that the initial remission was followed by recurrent tumor growth in most studies. Interestingly, different oncogenes controlled tumor growth in the recurrent than in the primary tumors. This could explain the astonishing clinical observation that inhibitors of a broader spectrum of protein kinases (so-called: "dirty inhibitors") may be superior over highly specific substances. Due to their additional "unspecific" inhibition of a broader spectrum of kinases, they may hamper the escape mechanisms by antagonizing also the pathways controlling recurrent tumor growth. (ii) Experiments with cell systems that allow switching-on oncogene expression point to a so far possibly underestimated cancer drug target: the dormant tumor cell. Oncogene expression (for instance: NeuT or RAS) led to a phenomenon named oncogene-induced senescence or dormancy. Dormant cells are unresponsive to mitogenic stimuli. Importantly, such cells are not at all ready to die, but can remain viable for extended periods of time. Recently, dormant tumor cells have been shown to be more resistant to stresses such as hypoxia or exposure to cytostatic drugs. It still is a matter of debate if and under which conditions dormant tumor cells can be "kissed to life". If these cells contribute to carcinogenesis, it will be important to identify substances specifically killing senescent cells. This review will focus on the possible relevance of senescence both as a pre-oncogenic condition and also for therapy.

RNAi knock-down mice: an emerging technology for post-genomic functional genetics.

RNA interference (RNAi) has been extensively used for sequence-specific silencing of gene function in mammalian cells. The latest major breakthrough in the application of RNAi technology came from experiments demonstrating RNAi-mediated gene repression in mice and rats. After more than two decades of functional mouse research aimed at developing and continuously improving transgenic and knock-out technology, the advent of RNAi knock-down mice represents a valuable new alternative for studying gene function in vivo. In this review we provide some basic insight as to how RNAi can induce gene silencing to then focus on recent findings concerning the applicability of RNAi for regulating gene function in the mouse. Reviewed topics will include delivery methods for RNAi-mediating molecules, a comparison between traditional knock-out and innovative transgenic RNAi technology and the generation of graded RNAi knock-down phenotypes. Apart from the exciting possibilities RNAi provides for studying gene function in mice, we discuss several caveats and limitations to be considered. Finally, we present prospective strategies as to how RNAi technology might be applied for generating conditional and tissue-restricted knock-down mice.

Synaptic vesicle alterations in rod photoreceptors of synaptophysin-deficient mice.

The abundance of the integral membrane protein synaptophysin in synaptic vesicles and its multiple possible functional contributions to transmitter exocytosis and synaptic vesicle formation stand in sharp contrast to the observed lack of defects in synaptophysin knockout mice. Assuming that deficiencies are compensated by the often coexpressed synaptophysin isoform synaptoporin, we now show that retinal rod photoreceptors, which do not synthesize synaptoporin either in wild-type or in knockout mice, are affected by the loss of synaptophysin. Multiple pale-appearing photoreceptors, as seen by electron microscopy, possess reduced cytoplasmic electron density, swollen mitochondria, an enlarged cell surface area, and, most importantly, a significantly reduced number of synaptic vesicles with an unusually bright interior. Quantification of the number of synaptic vesicles per unit area, not only in these, but also in all other rod terminals of knockout animals, reveals a considerable reduction in vesicles that is even more pronounced during the dark period, i.e., at times of highest synaptic activity. Moreover, activity-dependent reduction in synaptic vesicle diameter, typically occurring in wild-type mice, is not detected in knockout animals. The large number of clathrin-coated pits and vesicles in dark-adapted synaptophysin knockout mice is taken as an indication of compensatory usage of synaptophysin-independent pathway(s), and, conversely, in view of the overall reduction in the number of synaptic vesicles, as an indication for the presence of another synaptophysin-dependent synaptic vesicle recycling pathway. Our results provide in vivo evidence for the importance of the integral membrane protein synaptophysin for synaptic vesicle recycling and formation.

Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies.

In alcoholic hepatitis, a severe form of alcohol-induced toxic liver injury, as well as in experimental intoxication of mice with the porphyrinogenic drugs griseofulvin and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine, hepatocytes form cytoplasmic protein aggregates (Mallory bodies; MBs) containing cytokeratins (CKs) and non-CK components. Here we report that mice lacking the CK8 gene and hence CK intermediate filaments in hepatocytes, but still expressing the type I partner, ie, the CK18 gene, do not form MBs but suffer from extensive porphyria and progressive toxic liver damage, leading to the death of a considerable number of animals (7 of 12 during 12 weeks of intoxication). Our observations show that 1) in the absence of CK8 as well as in the situation of a relative excess of CK18 over CK8 no MBs are formed; 2) the loss of CK8 is not compensated by other type II CKs; and 3) porphyria and toxic liver damage are drastically enhanced in the absence of CK8. Our results point to a protective role of CKs in certain types of toxic liver injury and suggest that MBs by themselves are not harmful to hepatocytes but may be considered as a product of a novel defense mechanism in hepatocytes.

The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential.

Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C-domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so-called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.

Mice lacking synaptophysin reproduce and form typical synaptic vesicles.

Synaptophysin is one of the major integral membrane proteins of the small (30-50nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. The behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.

Identification of amino acid sequence motifs in desmocollin, a desmosomal glycoprotein, that are required for plakoglobin binding and plaque formation.

By transfecting epithelial cells with gene constructs encoding chimeric proteins of the transmembrane part of the gap junction protein connexin 32 in combination with various segments of the cytoplasmic part of the desmosomal cadherin desmocollin 1a, we have determined that a relatively short sequence element is necessary for the formation of desmosome-like plaques and for the specific anchorage of bundles of intermediate-sized filaments (IFs). Deletion of as little as the carboxyl-terminal 37 aa resulted in a lack of IF anchorage and binding of the plaque protein plakoglobin, as shown by immunolocalization and immunoprecipitation experiments. In addition, we show that the sequence requirements for the recruitment of desmoplakin, another desmosomal plaque protein, differ and that a short (10 aa) segment of the desmocollin 1a tail, located close to the plasma membrane, is also required for the binding of plakoglobin, as well as of desmoplakin, and also for IF anchorage. The importance of the carboxyl-terminal domain, homologous in diverse types of cadherins, is emphasized, as it must harbor, in a mutually exclusive pattern, the information for assembly of the IF-anchoring desmosomal plaque in desmocollins and for formation of the alpha-/beta-catenin- and vinculin-containing, actin filament-anchoring plaque in E- and N-cadherin.

Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage.

The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsg1 tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes.

Cytoskeletal architecture and epithelial differentiation: molecular determinants of cell interaction and cytoskeletal filament anchorage.

Desmosomes are morphologically well defined junctions between epithelial cells and also some other cells such as myocardiocytes, meningeal cells and dendritic reticulum cells of lymphatic follicles. Besides their function in cell coupling, desmosomes anchor components of the cytoskeleton, i.e. intermediate-sized filaments (IFs), through their cytoplasmic plaques, thereby contributing to cytoskeletal and tissue architecture. In molecular terms, desmosomes are specific assemblies of transmembrane glycoproteins of the cadherin family, desmoglein(s) and desmocollin(s), that contribute to cell adhesion via their extracellular, aminoterminal domains and to plaque formation and IF coupling through their cytoplasmic, carboxyterminal "tails". Using transfection assays, we analyzed the function of different tail domains in plaque assembly and IF anchorage. Furthermore, we present evidence that both desmogleins and desmocollins represent multigene subfamilies showing cell type specific expression and that a desmosomal plaque protein occurring in stratified and complex epithelia, the "band 6 protein", is related to the plakoglobin family.

Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage.

To examine the potential of cytoplasmic portions ("tails") of desmosomal cadherins for assembly of desmosome plaque structures and anchorage of intermediate filaments (IFs), we transfected cultured human A-431 carcinoma cells, abundant in desmosomes and cytokeratin IFs, with constructs encoding chimeric proteins in which the transmembranous region of connexin 32 had been fused with tails of desmocollin (Dsc) or desmoglein (Dsg). The results show that the tail of the long splice form a of Dsc, but not its shorter splice form b, contains sufficient information to recruit desmoplakin and plakoglobin to connexon membrane paracrystals (gap junctions) and to form a novel kind of plaque at which cytokeratin IFs attach. By contrast, chimeras containing a Dsg tail, which accumulated in the plasma membrane, showed a dominant-negative effect: they not only were unable to form gap junction structures and plaques but also led to the disappearance of all endogenous desmosomes and the detachment of IFs from the plasma membrane.

[Secretion of recombinant human epidermal growth factor into the periplasm in Escherichia coli cells]. / Sekretsiia v periplazmu rekombinantnogo épidermal'nogo faktora rosta cheloveka v kletkakh Escherichia coli.

Secretion vectors were constructed in which a synthetic gene of human epidermal growth factor (hEGF) joined with a gene coding for the leader peptide to one of the E. coli outer membrane major proteins (OmpF) is controlled by tac promoter. The increase of the hEGF yield was achieved by the multiplication of the gene copies. The hEGF in bacterial cells was secreted into periplasm. The recombinant protein was isolated by means of reverse phase chromatography as almost homogenous preparation (greater than 98%), the yield being 7 mg/l bacterial culture. The sequence of twenty-five N-terminal amino acid residues of the isolated hEGF coincided with that of the natural protein. The preparation proved to be biologically active.

[Biological effect of human erythropoietin in transgenic mice]. / Biologicheskii éffekt éritropoétina cheloveka u transgennykh myshei.

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).

[Introduction of DNA sequences of Rous sarcoma virus into Drosophila and mouse genomes by microinjections into ova]. / Vvedenie posledovatel'nostei DNK virusa sarkomy Rausa v genomy drosofily i myshi putem mikroin''ektsii v iaitsekletki.

The paper covers experimental results of introducing exogenic genetic material, namely DNA sequences of the Rous sarcoma virus, by microinjections in mice zygotes and Drosophila early embryos. In a number of cases integration of viral DNA into genomes of these organisms was detected. Blot-hybridizations analysis of cell DNA proved that the inserted viral sequences undergo rearrangements in the course of integration.

Microinjection of simian adenovirus SA7 (C-8) DNA into the mouse zygotes: differential distribution of viral DNA in organs.

A simian adenovirus SA7 (C-8) DNA was microinjected into fertilized mouse eggs. Thirty-five mice derived from eggs injected with SA7 DNA were screened for the presence of the adenovirus genome in their liver DNA. Eighteen of these mice contained the virus-specific sequences. SA7 DNA was detected in some tissues, but in all cases, viral sequences were absent from muscle and heart DNA. Viral DNA was inherited by 50-70% of the next generation. One mouse that contained about 1 copy of SA7 DNA per haploid genome has been shown to pass it on to five generations, although the integrated viral DNA sustained a considerable structural change between F1 and F3. RNA analysis in various organs of 12 mice has shown the transcription of SA7 DNA to be very infrequent: only in the kidney of one mouse and in the spleen of another did RNA contain SA7-specific sequences.

[Electron microscopic study of the intercellular junctions during the ciliary epithelial differentiation of the eye of the common frog]. / Elektronno-mikroskopicheskoe izuchenie mezhkletochnykh soedinenii pri differentsirovke tsiliarnogo épiteliia glaza travianoi liagushki.

Intercellular junctions in the eye ciliary epithelium were studied in Rana temporaria by means of transmission electron microscopy beginning from the stage of appearance of the light sensitivity in the larval eye and until the completion of metamorphosis. In the ciliary epithelium inner layer the apical parts of adjacent cells are tied by contacts forming the junction complex already at a very early developmental stage. The most apical position in this complex is occupied by the focal junction which appears to be an early stage of zonula occludens; this complex includes also zonula adherens and macula adherens which may follow in any succession focal junction or zonula occludens. No definite order was found in the localization of cell contacts between the outer and inner layers of ciliary epithelium, as well as between the side surfaces of the cells within each layer. Contacts were found everywhere termed as "lengthy" which may be considered as gap junctions. Regional differences were found in the ultrastructure of the ciliary epithelium cells with respect to unequal distribution of functional loads.